Abstract
Synaptic pruning is a fundamental process of neuronal circuit refinement in learning and memory. Accumulating evidence suggests that glia participates in sculpting the neuronal circuits through synapse engulfment. However, whether glial involvement in synaptic pruning has a role in memory formation remains elusive. Using newly developed phagocytosis reporter mice and three-dimensional ultrastructural characterization, we found that synaptic engulfment by cerebellar Bergmann glia (BG) frequently occurred upon cerebellum-dependent motor learning in mice. We observed increases in pre- and postsynaptic nibbling by BG along with a reduction in spine volume after learning. Pharmacological blockade of engulfment with Annexin V inhibited both the spine volume reduction and overnight improvement of motor adaptation. These results indicate that BG contribute to the refinement of the mature cerebellar cortical circuit through synaptic engulfment during motor learning.
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Data and resources are available on request from the corresponding authors. Source data are provided with this paper.
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Code used to present visual stimulation for this paper is available on request from the corresponding authors.
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Acknowledgements
This research was supported by JSPS KAKENHI grants 16K18388, 18K06457, 18J00603 (to Y.M.M.), 18H05110, 19H03338, 20H05046, 20H05896, 22H02713 (to K.M.),16H06280, 18H05120, 19K22469 and 19H03323 (to Y.F.) by The Naito Foundation Grant (to Y.M.M.), by the Satoshi Ohmura Foundation (to Y.M.M.), by Technical support platforms for promoting research of Advanced Bioimaging Support JP16H06280 (to Y.M.M. and N.Ohono.), by Opto-Science and Technology, the NOVARTIS Foundation, Takeda Science Foundation (to K.M), by The Joint Usage/Research Program of Medical Research Institute (to T.A., K.T. and K.M.), by AMED under the Brain/MINDS project: JP21dm0207111 (to H.H.) and by a Columbia University Target of Opportunity award. We thank T. Maegawa (Fukui University), A. Imai and N. Hattori (NIPS) for their helpful technical assistance in EM data acquisition, N. Hosoi, A. Konno (Gunma University), K. Chen (Columbia University), T. Osakada and L. Weinhard (New York University) for their helpful technical discussion, WB. Gan (Peking University Shenzhen Graduate School) for critical reading of the manuscript and all the members of the Matsui lab.
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Y.M.M. and K.M. conceived and designed the study. Y.M.M. performed most of the experiments and analysis. Y.F. performed FIB-SEM data acquisition. Y.N. and N.E. contributed to the FIB-SEM analysis. M.M. and N. Ohno. performed SBF-SEM data acquisition and analysis. Y.M.M, Y.F. and N.Ohno interpreted EM data. H.H. provided AAV. T.A., K.T., K.B., S.M., H.I., N. Osumi. and K.F.T. generated transgenic animals. E.S. and S.K. contributed gene-knockout experiments, G.Y. assisted in the experiments and H.I. wrote the HOKR program. Y.M.M. and K.M. wrote the manuscript. All authors discussed the results and commented on the manuscript.
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Nature Neuroscience thanks Won-Suk Chung, Cagla Eroglu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 No leak expression of pHRed in BGs.
a, Representative images of pHRed expression and PV-IHC in PV-tTA; tetO-pHRed mice (12 weeks old; 4 weeks after Dox off). Most PCs expressed pHRed. We have done 1 independent experiment (n = 3 mice). b, PV-tTA mouse was crossed with tetO-FRET-sensor (PV-Venus) mouse. This mouse was generated in the same way as the tetO-pHRed mouse. As with the pHRed in PV-pHRed mouse, Venus expression was observed in PCs and interneurons. Venus was not found in BGs, Iba1 positive microglia, and CD31 positive endothelial cells. This result suggests that the gene expression is induced selectively in neurons with PV-tTA. pHRed found in glia is due to internalization of pHRed into the glia. We have done 1 independent experiment (n = 2 mice). c, Red and green fluorescence merged images from WT, 4w Dox off, and no Dox of PV-pHRed mice (all with 3PGDH staining for BGs). Dashed square areas are enlarged in inset with BG outline shown with yellow line. Quantification of the red fluorescence signal is shown on the right. pHRed gene expression in PC and pHRed internalization into BG are increased after Dox off (n = 17, 25, 29, 37, 29 PCs from 4, 3, 4, 3, 3 mice; 35, 45, 52, 42, 30 BGs from 5, 3, 4, 5, 3 mice). One-way ANOVA followed by Dunnett’s multiple comparison test (F (4, 13) = 80.93, p < 0.0001; **** p < 0.0001 for PC; F(4, 15) = 10.51, p = 0.0003; * p = 0.02, *** p = 0.0004 for BG). d, Magnified images of the dashed white square in a (Arrowhead: BG soma; dashed white circles: pHRed signals in BG; Asterisk: PC soma). Right graph, line profiles of the normalized fluorescence intensities at the yellow line shown in the left panel image. pHRed signals are found, but no PV-IHC signals are found in BG soma. e, PC and BG soma images from a 12-week-old, 4 weeks off Dox PV-pHRed mouse. No Calbindin-IHC signals in BG. f, Images from a 6-week-old, no Dox PV-FRET mouse. Venus (GFP antibody)-IHC signals were seen only in PC soma and not in BG. g, No membrane GFP-IHC signal in BG in PV-tTA; tetO-LckE2GFP mice (12 weeks old, 4 weeks off Dox). h, Images from PV-tTA; tetO-pHRed; tetO-LckE2GFP trigenic mice (12 weeks old, 3 weeks off dox). Punctate pHRed signals were found in BG soma, but no GFP-IHC signals in BG. i, Representative images from WT, Mlc1-tTA; tetO-pHRed, PV-tTA; tetO-pHRed mice. Yellow dashed lines and white dashed lines demarcate PC and BG soma, respectively. pHRed signals were found in BGs of the Mlc1-pHRed mouse and in BGs of the PV-pHRed mouse, but pHRed signals were not found in PCs of the Mlc1-pHRed mouse. This shows that inter-cell material transfer is unidirectional between PC and BG (n = 19, 31, 23, 32 PCs, 28, 37, 23, 30 BGs from 4 mice). One-way ANOVA followed by Dunnett’s multiple comparison test (F (3, 12) = 26.46, p < 0.0001; **** p < 0.0001 for PC; F(3, 12) = 23.23, p < 0.0001; * p = 0.040, **** p < 0.0001 for BG; NS p > 0.05). Data are shown as mean ± s.e.m.
Extended Data Fig. 2 Engulfment of neuron-derived pHRed signal in microglia.
Representative images of microglial engulfment of pHRed in the cerebellar white matter of the PV-pHRed mouse. pHRed is expressed in Purkinje cell axons. Condensed pHRed signals are highlighted by dashed circles. These pHRed signals are contained in the microglial process and soma. We have done 1 independent experiments (n = 3 mice).
Extended Data Fig. 3 BG engulf Purkinje cell materials.
a, b, Purkinje cell specific promoter-tTA mediated pHRed expression. AAV2/9 L7-4-tTA was injected in the lobule IV/V of the tetO-pHRed mouse. pHRed expression was observed in the Purkinje cell layer (yellow arrowheads). pHRed expression was exclusive in PC (Asterisks in b; Calbindin: PC marker) with a punctate pHRed signal also found in BG (Arrowheads and dotted circle area: BG). We have done 2 independent experiments (n = 6 mice). c, d, AAV uninjected and injected lobules of the tetO-pHRed mice were compared. In the injected lobule, pHRed signals in BG were observed with higher intensity than the background (in WT and uninjected lobule; n = 23, 46, 31 BGs from 4 WT, 6 injected mice). One-way ANOVA followed by Tukey’s multiple comparison test (F (2, 13) = 20.59, p < 0.0001; *** p = 0.0005 for WT vs inj.; ***p = 0.0002 for uninj. vs inj.). Data are shown as mean ± s.e.m.
Extended Data Fig. 4 Reduction of mGluR1a signals after HOKR training.
a, Representative image of mGluR1a punctate signals in the molecular layer (ML) of the cerebellar cortex. b, High magnified images from Fig. 4f of the mGluR1a punctate signals localized on PC spines (Arrowheads: spine-like localization). VGLUT1 positive parallel fiber boutons appose these mGluR1a puncta. Arrowheads: spine-like localization. We have done more than 4 independent experiments. c, Cumulative frequencies of distal ML of mGluR1a IHC signal intensity. Significant reduction of the mGluR1a intensity was observed in HOKR trained mice (n = 6 mice). Kolmogorov-Smirnov test (**** P < 0.0001). d, The reduction of the mGluR1a immunostaining signal intensity after HOKR training was not dependent on the depth from the slice surface (n = 3 mice). Two-sided unpaired t-test (* p = 0.05, 0.037, 0.024, 0.023, 0.016, 0.011, 0.0012, 0.014 from 0 to 2.31 µm with 0.33 µm step). e, The intensity of mGluR1a in Pfl was not different between sham and HOKR trained mice (n = 6 mice). Two-sided unpaired t-test (p = 0.88). Data are shown mean ± s.e.m.
Extended Data Fig. 5 Phagocytic features in microglia were unchanged after HOKR training.
a, Sparse distribution of microglia (Iba1) in the ML (Sham, above) and no accumulation of microglia into Fl after HOKR training (below). b, Representative images of Iba1, PV-pHRed, and CD68 in ML with (sham) or without training (HOKR, 24hrs). c, No significant differences in the pHRed expression in PC, pHRed internalization into microglia (MG), CD68, and Iba1 expression level in ML without (sham) or with HOKR training (n = 67, 85 PCs, 14, 18 Microglia from 3 mice). Two-sided unpaired t-test (p = 0.65 for PC pHRed; p = 0.31 for MG pHRed; p = 0.97 for CD68; p = 0.53 for Iba1). Data are shown as mean ± s.e.m.
Extended Data Fig. 6 Definition of glial engulfment structure.
a, Representative image of glial engulfment structures of an axon and a spine head (same example as shown in Fig. 5d). Gray values from different ROIs demarcated by the yellow dashed circles are shown on the right. Using serial image reconstruction (not shown), each segmentation can be tracked to its origins. Two engulfed structures shown were tracked back to their axon and spine head origin, respectively. These engulfed invaginations did not contain organelles and were less electric dense compared to their origins. Although these engulfed structures are still in continuum with their origin, such character of lower electron density appearance suggests that these invaginations are on their way to become independent from their origins and are becoming less viable components. Gray values of randomly picked engulfed invagination with neither organelle nor lamellar bodies were quantified (middle panel, n = 15 invaginations from 2 HOKR mice). Two-sided paired t-test (**** p < 0.0001). The line profile of the yellow line in the left panel is shown on the right panel. The distance between the membrane surrounding the engulfed structure and the membrane of the engulfed structure was wide. This indicates that the normal tight-membrane interaction between two different cells is lost in the engulfment structure. b, Spinules appear to be different from engulfed structures. Spinules are short vesicular or long vermiform evaginations that emerge from neuronal compartments, which are enwrapped by other cells. Thus, basically, spinules have similar shapes with the engulfed structures. However, in spinules, the electron density of the origin and the leading protrusion of the spinule is similar. The membrane interaction between cells appears to be intact with a constant tight distance between the spinule and the surrounding membrane. We defined the structure shown on the upper pannel as spinule and the lower panel as engulfment, respectively. In this case, aberrant organelles were found in the structure inside of the invagination, and tight membrane interaction with its surrounding was lost. In the presented example, both the spinule and the engulfed structure originate from the same spine head. Right, original serial sections in 10 nm z-step. Data are shown as mean. We have done 1 independent experiment.
Extended Data Fig. 7 Multiple examples of neuronal nibbling by BG.
Multiple examples of the nibbling by BG of the pre-synaptic (a-e; blue), the post-synaptic (f-i; orange), and the inhibitory interneurons (j). a-c, Typical examples of empty (a), sparse lamellar (b), and multilamellar (c) phagosomes (arrowheads) and their origins (asterisks). d, Three other representative examples of unknown aberrant organelles (red dashed circles) were frequently observed at the origin of the engulfed protrusions. e, One example of an unknown organelle connected to the expanded ER at the origin of the phagosome. f-i, Typical examples of empty (f), sparse lamellar (g), and multilamellar (h) phagosomes (arrowheads) and their origins (asterisks). i, Examples of aberrant lamellar structures in phagosomes connected to ERs in spines. j, Two other cases of the multilamellar body from herniated mitochondria (purple). All scale bars were 0.5 µm and serial images were taken at 50 nm z-steps. We have done 1 independent experiment.
Extended Data Fig. 8 mGluR1a IHC signal reduction after HOKR training was inhibited by Annexin V.
a, Representative images of Calbindin and mGluR1a IHC signals in sham, in HOKR with PBS, and in HOKR with Annexin V. mGluR1a punctate likely reflects the postsynaptic spine location. With HOKR training, mGluR1a signal decreased. Such decrease was not observed after HOKR training when Annexin V was treated to inhibit phagocytosis. We have done 3 independent experiments. b, Reduction of mGluR1a IHC signals in the flocculus (Fl) after HOKR training was inhibited with Annexin V treatment. Reduction of mGluR1a signal was not observed with HOKR training in the paraflocculus (PFl) either with PBS or with Annexin V treatment. Each dot shows data from a single animal (n = 4–10). One-way ANOVA followed by Bonferroni multiple comparison test (F (3, 20) = 2.49, p = 0.09; *p = 0.038 for PBS sham vs HOKR; p = 0.92 for Annexin V sham vs HOKR in Fl; F (3, 20) = 0.94, p = 0.44; p = 0.48 for PBS sham vs HOKR; p = 0.89 for Annexin V sham vs HOKR in PFl). Data are shown as mean ± s.e.m.
Extended Data Fig. 9 Enhancement of PS exposure in the Fl after HOKR learning.
a, PBS or Annexin V was intracisternally injected (iCM) and HOKR training was performed in a subset of animals. Annexin V was detected by immunohistochemical (IHC) staining of brain samples taken at 24 hours after the initial HOKR training on Day 1. Annexin V antibody is expected to react both to the Annexin V instrinsic to mouse and to the externally injected human recombinant Annexin V. Shown in this panel is representative images of IHC of Annexin V and Calbindin, which is expressed in PC dendrites and spines. This sample is from a HOKR trained mouse injected with Annexin V. Shown is the molecular layer of the flocculus (Fl). Numerous Annexin V signals could be localized at the proximity of dendrite-spine like structures. We have done 3 independent experiments. b, Annexin V IHC labeling was the most evident in the group of mice that was injected with Annexin V and had HOKR training. c, Quantification of Annexin V IHC signal in Fl (top) and PFl (bottom). PS exposure specifically occurred in the Fl after HOKR training and not in the PFl. Each dot shows single animal (n = 4–10 mice). One-way ANOVA followed by Bonferroni multiple comparison test (F (3, 18) = 12.98, p < 0.0001; p > 0.99 for PBS sham vs HOKR; ***p = 0.001 for Annexin V sham vs HOKR in Fl; F(3, 18) = 2.43, p = 0.09; p = 0.75 for PBS sham vs HOKR; p = 0.84 for Annexin V sham vs HOKR in PFl). Data are shown as mean ± s.e.m. Data are shown as mean ± s.e.m.
Extended Data Fig. 10 Bergmann glial ABCA1 mediate phagocytosis and HOKR learning.
a-d, Data from Tabula Muris. a, t-Distributed Stochastic Neighbor Embedding (t-SNE) plot of individual non-myeloid cell in the brain. Colors correspond to each cell type. b, c, Abca1 was enriched mainly in oligodendrocyte precursor cell, BG, and astrocytes. d, BG expressed multiple type of PS-dependent phagocytic pathway molecules including Abca1, Megf10, Lrp1, Mertk, Bai1, Itgav, Itgb5, as well as astrocytes markers. These features were similar to the general astrocyte population. e, Immunohistochemical detection of ABCA1 in the cerebellum. ABCA1 staining showed a similar morphological pattern of BG soma and processes (arrowheads). Scale bar: 100 µm. We have done 1 independent experiment. f, Schema for conditional targeting Abca1flox/flox with human GFAP-Cre. g, Protein quantification of ABCA1 by western blotting from the cerebellum. Conditional knockout (cKO) of Abca1 gene decreased ABCA1 protein in the cerebellar tissue (n = 2 mice). We have done 2 independent experiments. h, Schema of the experimental design (top left). AAV2/9-L7-4mCMV-tTA injected into tetO-pHRed; Abca1flox/flox with or without hGFAP-Cre mice to create PCs-specific pHRed expression in ABCA1 cKO mice. Frequency of finding of pHRed puncta within BG soma was apparently less in ABCA1 cKO mice (bottom left). pHRed intensity in BG was significantly less in ABCA1 cKO (middle graph) and no such difference between the control was observed for pHRed intensity in PC (right graph), (n = 3 mice). Two-sided unpaired t-test (p = 0.0051 for BG; p = 0.75 for PC). i, Schema for conditional targeting Abca1flox/flox with GLAST-CreERT2 mice (above) and the experimental schedule (below). Tamoxifen was injected at 8 weeks for consecutive 5 days. HOKR experiments were done after three weeks of wait time to achieve efficient KO of ABCA1. j, HOKR learning curves of three types of control groups and the cKO group are shown (For control: n = 9 (Abca1fl/fl: n = 5; Abca1fl/fl + Tam: n = 2; Abca1 fl/fl, Glast-CreERT2 n = 2) For ABCA1 cKO: n = 3 mice (Abca1fl/fl, Glast-CreERT2 + Tam). k, Significant improvement in the HOKR gain was observed from the 4th training session on Day 1 to the test session on Day 2 for the control group. However, such improvement was absent in the ABCA1 cKO group (control: n = 9; ABCA1 cKO n = 3 mice). Two-sided paired t-test (4th session vs 2nd day: p = 0.0081 for control; p = 0.072 for ABCA1 cKO). Unpaired t-test (p = 0.011 control vs ABCA1 cKO). All data are shown as mean ± s.e.m.
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Supplementary Figs. 1–4.
Supplementary Video 1
Eye movement before and after HOKR spaced learning. Representative examples of horizontal eye movements during the first learning session on the first day and test trial on the second day. The videos were captured by an infrared-sensitive CCD camera and the example eye movements were extracted by DeepLabCut (color dots).
Supplementary Video 2
FIB-SEM image sequences of engulfment by BG. FIB-SEM image sequences of engulfment of pre- and postsynapses by BG are shown. Cerebellum samples were taken from the HOKR-trained mouse. Original images and segmented image sequences are shown along with three orthogonal views of the same sample in Fig. 5a–c.
Supplementary Video 3
FIB-SEM 3D reconstruction of the engulfment. Using data from the same sample as in Fig. 5a–c and Supplementary Video 2, an FIB-SEM 3D reconstruction of the engulfment of pre- and postsynapses by BG in a HOKR-trained mouse cerebellum was created. In the latter part of the video of the 3D reconstruction, the engulfed structures are highlighted as nontransparent (solid) protrusions surrounded by the BG processes (magenta).
Supplementary Video 4
FIB-SEM 3D reconstruction of dendritic spines and engulfed protrusions. Representative 3D-reconstructed single–PC dendrite with spines (yellow) in a HOKR-trained mouse. Engulfed protrusions are highlighted in red.
Supplementary Video 5
FIB-SEM image sequences of representative engulfment from various compartment origins. Original FIB-SEM image sequences of the structures shown in Fig. 5d. Representative examples of engulfment of the axon, the bouton, the spine head, the spine neck and the dendrite are shown. On the left are the original FIB-SEM images and on the right are the same images overlaid with segmentation shown in semi transparent colors. Engulfed protrusions are highlighted with red arrows. All scale bars are 500 nm.
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Morizawa, Y.M., Matsumoto, M., Nakashima, Y. et al. Synaptic pruning through glial synapse engulfment upon motor learning. Nat Neurosci 25, 1458–1469 (2022). https://doi.org/10.1038/s41593-022-01184-5
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DOI: https://doi.org/10.1038/s41593-022-01184-5
